Teresa Fernández

Study of an Argentine Mistletoe, the hemiparasite Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae)

Ligaria cuneifolia (R. et P.) Tiegh. is an hemiparasite species used in Argentine folk medicine as a substitute for the European mistletoe (Viscum album L.) based on its putative activity of decreasing high blood pressure. This paper analyzes flavonoid composition, protein constituents and the possible immunomodulatory and antitumoral effects of this species. Micromolecular study disclosed quercetin-free, quercetin-glycosylated and proanthocyanidins corresponding to cyanidin monomers, which implies a particular metabolic pathway. Proteins present in L. cuneifolia extracts analyzed by SDS-PAGE presented multiple bands with molecular weights ranging from 14 to 90 kD. These features contribute to the characterization of the native mistletoe. As V. album is being used in cancer treatment due to its immunomodulatory and antitumoral activity, the action of aqueous L. cuneifolia extracts on murine lymphocytes was investigated. Culture of murine spleen cells alone or stimulated with Concanavalin A or lipopolysaccharide in presence of L. cuneifolia extracts indicated a certain stimulation of splenocytes alone and an inhibition of splenocytes stimulated with Concanvalin A or lipopolysaccharide. An inhibitory effect was also observed on the proliferation of murine leukemia cells. In addition, aqueous extracts increased nitric oxide production by murine macrophages. These results suggest that L. cuneifolia extracts exert an immunomodulatory effect on the mouse immune system.

Immunomodulating properties of Argentine plants with ethnomedicinal use.

Five Argentine medicinal plants selected according to folk traditional or ethnomedical use, references and primary pharmacological screening; were chosen to elucidate their immunomodulating properties. Dichloromethane, methanolic and aqueous extracts of the aerial parts of Achyrocline flaccida (A. flaccida), Eupatorium arnottianum (E. arnottianum) and Eupatorioum buniifolium (E. buniifolium), leaves of Lithraea molleoides (L. molleoides) and leaves and stems of Phyllanthus sellowianus (P. sellowianus) were analyzed to disclose their effects on murine normal and tumor cell growth as well as on complement hemolytic activity. Modulation of cell growth was evaluated by tritiated thymidine incorporation while inhibition of complement activity was measured on both classical and alternative complement pathways (CP and AP respectively). The results obtained show that most of the extracts exerted inhibitory effect on tumor as well as on mitogen activated normal spleen cell growth. On tumor cells, IC50 ranged between 1-75 microg/ml for most of the extracts with the exception of dichloromethane of L. molleoides and P. sellowianus which required concentrations higher than 100 microg/ml to produce the effect. On mitogenic activated splenocytes, IC50 ranged between < 1 to 85 microg/ml with the exception of methanolic extract of E. buniifolium or P. sellowianus which were not effective on ConA or LPS stimulated splenocytes respectively. Only E. buniifolium was active on murine normal splenocytes proliferation (IC50 0.5-1.5 microg/ml). Finally, one (7%) of 15 extracts showed inhibition of complement activity on CP and 6 extracts (40%) presented moderate activity on CP. The dichloromethane extract of E. arnottianum was the most active (IC50 5 microg/ml), although remarkable effect was also obtained with dichloromethane and methanolic extracts of P. sellowianus (IC50 11.2 and 17.3 microg/ml respectively). Besides, 2 extracts (13%), dichloromethane extract of E. arnottianum and aqueous extract of P. sellowianus, showed moderate inhibition on AP.

THE IMMUNOCHEMICAL REACTIVITY AND NEUTRALIZING CAPACITY OF POLYVALENT Vipera (EUROPEAN) ANTIVENOM ON ENZYMATIC AND TOXIC ACTIVITIES IN THE VENOMS OF CROTALIDS FROM ARGENTINA

The immunochemical reactivity and neutralizing capacity of polyvalent Vipera antivenom (Vipera ammodytes, Vipera aspis, Vipera berus, Vipera lebetina, and Vipera xanthina) were tested on the enzymatic and biological activities of Crotalus durissus terrificus and the following Bothrops venoms from Argentina (Bothrops alternatus, Bothrops ammodytoides, Bothrops neuwiedii, Bothrops jararaca, Bothrops jararacussu, and Bothrops moojeni). The Vipera antivenom reacted weakly when tested by double immunoprecipitation (DIP) and reacted with all the venoms when tested by ELISA. Several components in all the venoms studied were recognized in Western blots. Vipera antivenom deactivated to different degrees in vitro procoagulant, (indirect) hemolytic, and proteolytic activities in all the venoms studied. Preincubation of Bothrops alternatus venom with Vipera antivenom neutralized a lethal potency of 4.5 LD50 in mice with an ED50 of 1.25 ± 0.25 ml per mg of venom, and with 1.0 ml/mg inhibited 54% of the hemorragic activity and 48% of necrotic activity. Vipera antivenom (2.0 ml per mg toxin) inhibited the phospholipase A2 activity of purified crotoxin and decreased its lethal potency by 60%, while the neutralizing capacity on the lethal potency of crude Crotalus durissus terrificus venom was poor even at a level of 5.0 ml/mg of venom.

Some toxic and enzymatic activities of Bothrops ammodytoides (‘yarará ñata’) venom

Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom.

Screening of Anticancer and Immunomodulatory Activities of Panamanian Plants

Fifty-one extracts, from six Panamanian plants species selected on the basis of their toxicities against Artemia salina, belonging to five families and five genera, were screened for their in vitro anticancer (TK-10, UACC-62, MCF-7) and immunomodulatory activities [inhibition of complement, classical (CP) and alternative (AP) complement pathway and lymphoproliferation]. The results showed that 9 (17.6%) of 51 extracts presented anticancer activity against cancer cell lines, while 28 (54.9%) of 51 extracts stimulated normal spleen cells proliferation, and 25 (49.0%) inhibited in vitro lymphoid tumor cell growth. Finally, 7 (19.4%) and 10 (27.8%) out of 36 extracts showed high and moderate anticomplementary activity on CP, respectively. Moreover, out of 36 extracts, 1 (2.8%) produced higher inhibition while 4 (11.1%) showed moderate inhibition in the alternate pathway. The dichloromethane, methanolic and aqueous extracts from the leaf (3.0, 3.2, 4.2) and branch (3.5, 5.7, 5.8) of Trichospermum galeottii and from the leaf (2.4, 2.5, 5.6) and stem (2.3, 2.3, 4.7) of Sauraria yasicae were the most active immunomodulatory plants. The dichloromethane extract from the root of Morinda panamensis (8.7, 4.4, 6.3) was active against the three cancer cell lines, while the dichloromethane extract from the bark of Trichospermum galeottii(TK-10 = 3.4,UACC-62 = 9.4) and the bark of Morinda panamensis (UACC-62 = 5.5, MCF-7 = 5.9) as well as the methanolic extract from the root of Morinda rojoc (UACC-62 = 9.2, MCF-7 = 9.4) exhibited selectivity against two cancer cell lines.